EP0681695B1 - Appareil d'imagerie quantitative de fluorogenes multiples - Google Patents

Appareil d'imagerie quantitative de fluorogenes multiples Download PDF

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EP0681695B1
EP0681695B1 EP94906355A EP94906355A EP0681695B1 EP 0681695 B1 EP0681695 B1 EP 0681695B1 EP 94906355 A EP94906355 A EP 94906355A EP 94906355 A EP94906355 A EP 94906355A EP 0681695 B1 EP0681695 B1 EP 0681695B1
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beams
amplifier
targets
fluorescent
phase
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EP0681695A1 (fr
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Nils R. D. Aslund
Kjell S. Carlsson
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/645—Specially adapted constructive features of fluorimeters
    • G01N21/6452—Individual samples arranged in a regular 2D-array, e.g. multiwell plates
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6408—Fluorescence; Phosphorescence with measurement of decay time, time resolved fluorescence
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N2021/6417—Spectrofluorimetric devices
    • G01N2021/6419—Excitation at two or more wavelengths
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N2021/6417—Spectrofluorimetric devices
    • G01N2021/6423—Spectral mapping, video display
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G01N2021/6439—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks
    • G01N2021/6441—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks with two or more labels
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/645—Specially adapted constructive features of fluorimeters
    • G01N21/6456—Spatial resolved fluorescence measurements; Imaging

Definitions

  • This invention relates to quantitative microfluorometry and more particularly to a device for quantitative microfluorometry in which fluorophores are quantified by simultaneous excitation at two or more wavelengths.
  • the emitted fluorescent radiation from a fluorophore in response to excitation of a single wavelength generally includes a broad band of wavelengths. Spectra of emissions from different fluorophores will generally overlap. Also, when using radiations of different wavelengths to excite each of two fluorophores, their emission spectra may still overlap.
  • Fluorophores absorb excitation radiation at more than one wavelength.
  • the absorption spectra of different fluorophores may overlap.
  • radiation of a wavelength chosen to efficiently excite a certain fluorophore will also to some extent excite other fluorophores.
  • the fact that their emission spectra generally overlap and that their absorption spectra also may overlap constitutes a major problem.
  • simultaneous fluorescent detection has been primarily limited to cases where the wavelength regions of spectral overlap of the emissions can be suppressed by optical filtering. Then separation is achieved at the expense of losing valuable signal intensity.
  • the different emission spectra of a single fluorophore obtained by exciting it with different wavelengths generally overlap heavily and can not be separated by optical filtering.
  • the relative intensities of these spectra depend on the shape of the absorption spectrum. This shape may convey valuable information about the fluorophore and its environment.
  • the individual contributions from each fluorophore must be measured separately. This can not be done by optical filtering exclusively.
  • a light waveform measuring device is used for measuring the lifetime of fluorescent light produced due to pulsed laser excitation.
  • Two laser beams of different frequencies, at least one of which is pulsed, are used to produce a single-frequency pulsed beam selected from the sum frequency mixing of the beams.
  • the output beam is pulsed at the same rate as the pulsed input beam which is used to trigger a single-photon detector or streak camera. The detector is thereby synchronized to the exciting beam.
  • the prior art devices which measure fluorescence at only one peak emission wavelength are unable to simultaneously quantify multiple fluorophores using the total emission from each fluorophore.
  • Those devices which rely on spectral separation to distinguish multiple fluorophores are unable to separate the total contribution of each fluorophore from the combined emission spectrum detected.
  • phase angle can be derived from the outputs of the in-phase and the quadrature channels of the amplifier. This has been used, for example, in cytometry by Steinkamp and Crissman (1992) to distinguish between fluorophores that have different decay times. It has also been proposed by Morgan et al. (1992) that this technique should be used in a confocal scanning microscope to produce images that represent the decay time measured at each picture point. A non-confocal system that records the decay times in all picture elements simultaneously has been implemented by Lakowicz and Berndt (1991).
  • Another method to distinguish between fluorophores with different decay times is to use a repetitive source of short optical pulses and employ time-correlated single-photon counting. This technique has also been combined with imaging.
  • a non-confocal system of this kind has been implemented by Morgan and Murray (1991).
  • a confocal system using time gating has been implemented by Buurman et al. (1992).
  • FLIM fluorescence lifetime imaging
  • each excitation wavelength is chosen to predominantly excite one of the fluorophores.
  • the intensity of the excitation at each wavelength is time-modulated at a separate frequency.
  • the radiation emitted in response to the combined excitation is separated into spectral parts representing different wavelength bands.
  • Optical filtering and optical beam splitting is employed to achieve this separation.
  • Dual detectors are used to detect the separated spectral parts of the combined emission.
  • a frequency-locked amplifier synchronized to a corresponding modulation frequency is attached to each detector. The discrimination performed by the frequency-locked amplifiers, in combination with the separation into spectral parts performed by optical filters and a beam splitter, allows extraction and measuring of contributions representing each separate fluorophore exclusively.
  • two single-phase lock-in amplifiers are attached to each detector.
  • the two amplifiers are synchronized with different frequencies, viz. the frequencies used to modulate the intensity of the excitation at each of two excitation wavelengths. All the individual components of the composed emission, including cross-excited contributions, are extracted and measured.
  • two single-phase amplifiers attached to each detector are used, the two amplifiers being tuned to the same frequency.
  • the two amplifiers attached to the one detector are both tuned to the frequency used to modulate the intensity of the excitation at the one excitation wavelength; the two amplifiers attached to the other detector are both tuned to the frequency used to modulate the intensity of the excitation at the other excitation wavelength.
  • the phase positions of the two amplifiers attached to each detector are 90° apart. Together the two amplifiers perform the function of a dual-phase lock-in amplifier encompassing an in-line and a quadrature channel.
  • the two outputs from each dual-phase amplifier are processed to obtain the magnitude and the phase angle of the signal applied to the amplifier, employing well-known techniques. Preferably this is performed digitally, by a data processing unit attached to the outputs. As a result the amplitude of the modulated signal received by the detector, and a phase angle, are derived.
  • the phase angle represents, in a relative scale, the shift between the phase angle of the modulated excitation and the phase angle of the detected emission. If the molecular relaxation time of a fluorophore changes, as it may do, e.g., due to a change of the pH-value of the environment of the fluorophore, this phase shift will change. The effect is substantial if the modulation frequency is high. Therefore, in the present variation of the first embodiment, the modulation frequency used is preferably an order of magnitude higher than in the previously described variations.
  • the device according to the described variation supplies, simultaneously and independently, the phase shifts for two different fluorophores.
  • confocal scanning optics may be used for pixel-by-pixel scanning of a stack of planes, so that volumetric data may be obtained, simultaneously and independently, from different fluorophores.
  • Data may be stored in a computer and later displayed.
  • Different colors may be used to distinguish regions representing different intensities of a fluorophore or, in one of the variations of the first embodiment, different decay times of a fluorophore. In the latter case, a FLIM image is obtained.
  • colors may be used to distinguish contributions from different fluorophores.
  • a three-dimensional image of the volume may be re-created and closely inspected using image-processing techniques, such as generating projections from different angles, zooming etc.
  • a single detector is used. This embodiment has relevance in cases where the relative changes in relaxation times are negligible.
  • a separate lock-in amplifier is synchronized to the modulation frequency of each excitation wavelength.
  • dual phase lock-in amplifiers are used with the in-phase channel tuned to a phase position near to that of the fluorophore predominantly excited by that wavelength. Using the in-phase and quadrature outputs of each amplifier, all components of the combined emission spectrum may be separated.
  • phase angles of the emission spectra are constants, since the relaxation times do not change, and are determined from data obtained in regions where only one of the fluorophores is present. These regions may be identified by direct observation or by analysis of the data set from the entire specimen.
  • the described variation of the second embodiment is thus identical to a variation of the first embodiment where two single-phase amplifiers are attached to each detector and the two amplifiers attached to each detector are both tuned to the same frequency.
  • a single phase lock-in amplifier is used for each excitation wavelength.
  • the phase position of the amplifier is tuned to the position that gives a maximum output value for the fluorescence emitted by the predominantly excited fluorophore.
  • the emission spectra corresponding to each predominantly excited fluorophore may be discriminated.
  • microfluorometer An advantage of the microfluorometer is that multiple fluorophores are simultaneously and independently quantified with greater efficiency.
  • Another advantage is that contributions from different fluorophores are measured with negligible loss of information, despite their emission spectra overlap and despite interference from cross-excited spectra.
  • Another advantage is that all the contributions from each fluorophore, including cross-excited spectra, may be simultaneously measured.
  • a further advantage is that fluorescent lifetime images from different fluorophores are simultaneously recorded.
  • Fig. 1 is a schematic block diagram of a dual detector quantitative microfluorometer in accordance with our invention.
  • Figs. 2a-d are plots showing the individual spectral components of a combined emission spectrum.
  • Fig. 3 is a schematic block diagram of a first variation of the device of Fig. 1 featuring dual lock-in amplifiers for each detector.
  • Fig. 4 is a schematic block diagram of a second variation of the device of Fig. 1 featuring dual lock-in amplifiers for each detector configured for phase discrimination as well as frequency discrimination.
  • Fig. 5 is a schematic block diagram of a single detector quantitative microfluorometer in accordance with the present invention.
  • Figs. 6a-d are plots showing the individual spectral components of a combined emission spectrum.
  • Figs. 7a-b are a diagrammatic representation of the phase relation between the modulated excitation wavelength and the modulated fluorescent emissions from two fluorophores for each of the lock-in amplifiers.
  • Fig. 8 is a vector diagram of the phase angles of the components of the output of dual phase lock-in amplifier 1.
  • Fig. 9 is a vector diagram showing the phase relationship of the components of the output of dual phase lock-in amplifier 2.
  • Fig. 10 is a schematic block diagram of a variation of the second embodiment of the present invention using single phase lock-in amplifiers.
  • a schematic block diagram of the first embodiment of the quantitative microfluorometer employing two excitation wavelengths, two detectors and two lock-in amplifiers is shown.
  • Monochromatic excitation light of different wavelengths is supplied by a pair of light sources 12 and 14.
  • light sources 12 and 14 are separate diode lasers, although any source of electromagnetic radiation capable of being time-modulated in intensity may be used.
  • the intensity of light source 12 is controlled by a modulator 16 operating at a sinusoidal frequency ⁇ 1 .
  • light source 14 is controlled by a second modulator 18 operating at frequency ⁇ 2 .
  • the combination of light source 12 and modulator 16 may alternatively be implemented by a continuous gas laser, followed by an electro-optical or an acousto-optical modulator operating at a frequency ⁇ 1 .
  • the combination of light source 14 and modulator 18 may be implemented by a continuous gas laser, followed by an electro-optical or acousto-optical modulator operating at a frequency ⁇ 2 .
  • these modulators should be capable of operating at frequencies up to 50 Mhz to cover phase shift measurements according to one of the preferred embodiments. Realistic values of the modulation frequencies in the other embodiments are 2.2 and 3.0 Mhz. Individual light beams from light sources 12 and 14 are combined in a beam combiner 20.
  • the combined beam passes through a beamsplitter 22, which transmits light at the excitation wavelengths ⁇ 1 and ⁇ 2 .
  • a scanner 24 sequentially directs the combined beam to locations at sample 26.
  • a realistic value of the sampling frequency of the scanner is 100 khz.
  • scanner 24 is a confocal laser microscope, although any device capable of focusing excitation at, and collecting emitted radiation from, a discrete location in sample 26 may be used.
  • Sample 26 may be any light-transmitting object containing multiple fluorophores. Such objects include biological cells and two dimensional gels used for separating compounds or dish cultures and the like.
  • the combined fluorescence emitted in response to excitation at the focal point of scanner 24 is collected by scanner 24 and directed to beamsplitter 22, which has a dichroic element, then passed through another beamsplitter 102, which deflects emitted fluorescence to the two detectors 114 and 116.
  • Detectors 114 and 116 may each be any device which produces a signal in response to the photons emitted by the fluorophores. Photomultipliers which convert photons into electrical signals are examples of such devices.
  • the semi-reflecting element of the beamsplitter 102 may be a dichroic mirror, performing both beamsplitting and spectral separation.
  • optical filters 104 and 106 may be employed.
  • the effect of the optical filtering performed either by a dichroic element included in beamsplitter 102 or by optical filters 104 and 106, or jointly by these elements, is to separate the collected light into two parts representing different wavelength bands. These parts are distributed to detectors 114 and 116, respectively. Detectors 114 and 116 may be any detectors which produce an electrical signal in response to photons emitted by the fluorophores. Photomultipliers, which convert photons into electrical signals, are examples of such devices.
  • the signals produced by detectors 114 and 116 are sent to a pair of lock-in amplifiers 30 and 32.
  • Lock-in amplifier 30 is synchronized with the modulation frequency ⁇ 1 imposed on light source 12 by modulator 16.
  • lock-in amplifier 32 is synchronized with the modulation frequency ⁇ 2 imposed on light source 14 by modulator 18. The phase position of each lock-in amplifier is adjusted to maximize the output.
  • the curves show the individual spectral components of a combined emission spectrum emitted by two fluorophores in response to excitation by two wavelengths.
  • the curves approximately describe the spectra of two commonly used fluorophores, TRITC (tetramethyl rhodamine isothiocyanate) (fluorophore A) and Lucifer Yellow (fluorophore B).
  • TRITC tetramethyl rhodamine isothiocyanate
  • fluorophore A tetramethyl rhodamine isothiocyanate
  • Lucifer Yellow fluorophore B
  • Fluorophore A produces a fluorescent emission spectrum 46 (I A ) in response to ⁇ 1 .
  • Fluorophore B produces a fluorescent emission spectrum 50 (I B ) in response to ⁇ 2 and a smaller fluorescent emission spectrum 52 (I b ) in response to ⁇ 1 .
  • This latter cross-excited spectrum results from the fact that absorption spectra 42 and 44 overlap at the wavelength ⁇ 1 .
  • the combined emission spectrum, in response to excitation by ⁇ 1 and ⁇ 2 is composed of the superimposed spectra 46 (I A ), 50 (I B ) and 52 (I b ), as indicated by Fig. 4d.
  • the contribution 50 (I B ) is the only of the three emissions that is modulated with the frequency ⁇ 2 .
  • the wavelength band 62 (b) deflected to detector 116 (detector 2) is chosen so as to encompass the major part of the spectral range of contribution 50 (I B ).
  • the measured value of the contribution I B is then obtained from the output of lock-in amplifier 32.
  • the contributions 46 (I A ) and 52 (I b ) are both modulated with frequency ⁇ 1 .
  • the wavelength band 60 (a) deflected to detector 114 (detector 1) is chosen so as to exclude the spectral range of contribution 52 (I b ) but to encompass the major part of contribution 46 (I A ).
  • the measured value of the contribution I A is then obtained as an output from lock-in amplifier 30.
  • the outputs from lock-in amplifiers 30 and 32 are sent to a computer 34.
  • computer 34 is a digital computer and the outputs from amplifiers 30 and 32 are digitized, before storage, by conventional analog to digital converters.
  • the inputs representing contributions I A and I B at discrete locations in sample 26 are stored in a location specific manner in the computer 34 and can be displayed as digital images on a screen.
  • An image representing fluorophore A or an image representing fluorophore B can be displayed separately.
  • an image representing the two fluorophores can be displayed simultaneously, e.g., using colors to distinguish them from each other.
  • Computer 34 may also derive projection images of a stack of recorded images representing a three-dimensional transparent volume, such as a cell, marked by two different fluorophores, and display such projection images in a similar fashion on a screen.
  • a stack of recorded images representing a three-dimensional transparent volume, such as a cell, marked by two different fluorophores
  • displayed data representing measured values of I A and I B are mapped into digital images 72 and 78 for visualization.
  • two lock-in amplifiers 122 and 124 have been provided for detector 114 (detector 1), and two lock-in amplifiers 126 and 128 for detector 116 (detector 2).
  • Lock-in amplifiers 122 and 126 are locked to modulator 16 (modulator 1), and lock-in amplifiers 124 and 128 to modulator 18 (modulator 2).
  • the phase position of each lock-in amplifier is adjusted to maximize the output.
  • the wavelength band deflected to detector 116 is chosen so as to encompass the major part of the spectral range of contribution 50 (I B ). It will also encompass major parts of the cross-excited contribution 52 (I b ).
  • the contribution 50 (I B ) is modulated with frequency ⁇ 2
  • contribution 52 (I b ) is modulated with frequency ⁇ 1 .
  • Lock-in amplifier 126 is locked to frequency ⁇ 1 and lock-in amplifier 128 to frequency ⁇ 2 .
  • the measured value of the contribution 50 (I B ) is then obtained as an output from lock-in amplifier 128, separated from any of the other contributions, i.e., separated from contribution 52 (I b ) and from contribution 46 (I A ), which are both modulated with frequency ⁇ 1 .
  • the measured value of the contribution 52 (I b ) is obtained as an output from lock-in amplifier 126, separated from contribution 50 (I B ), which is modulated with the frequency ⁇ 2 , and possibly with some interference from contribution 46 (I A ), the spectral range of which lies essentially in the wavelength band deflected to detector 114 (detector 1). If this interference is not negligible, it can be compensated for, as will be explained below.
  • the wavelength band deflected to detector 114 (detector 1) is chosen so as to exclude the spectral range of contribution 52 (I b ) but to encompass the major part of contribution 46 (I A ), modulated with frequency ⁇ 1 .
  • the measured value of the contribution 46 (I A ) is obtained as an output from lock-in amplifier 122, which is tuned to frequency ⁇ 1 , separated from all other a contributions, including contribution 50 (I B ), which is modulated with the frequency ⁇ 2 .
  • the measured value of the contribution 46 (I A ) does not include the minor part of that contribution which is deflected to detector 116 (detector 2).
  • this minor part which by an estimable amount is proportional to the measured part deflected to detector 114 (detector 1), is not negligible, it can be obtained directly as a proportion of the measured part of contribution 46 (I A ) obtained from detector 114 (detector 1).
  • measured values from detector 116 (detector 2) can be corrected for the interference from contribution 46 (I A ) by subtracting this proportion. This operation is preferably performed by computer 34.
  • lock-in amplifier 124 tuned to frequency ⁇ 2 , is to provide an output which is a measured value of a possible contribution I a , representing a cross-excited contribution from fluorophore A excited by wavelength ⁇ 2 .
  • This contribution will occur if absorption spectra 42 and 44 overlap also at wavelength ⁇ 2 .
  • the main part of the contribution I a if occurring, is located within the spectral range 60 (a) passed to detector 114.
  • the outputs from lock-in amplifiers 122, 124, 126 and 128 are sent to a computer 34.
  • the inputs to computer 34 representing contributions I A , I a , I b and I B , respectively, at discrete locations in sample 26 are stored in a location specific manner in the computer 34 and can be displayed as digital images on a screen. As explained in the description of the previous embodiment, they can be displayed separately or in combinations and in projection images from a volume.
  • quotient images can be presented, representing I a /I A or I b /I B , conveying information about the shape of the excitation spectra of fluorophores A and B, respectively.
  • stored data representing measured values of I A , I a , I b and I B are mapped into digital images 72, 74, 76 and 78 for visualization.
  • FIG. 4 A variation of the apparatus of Fig. 3 is shown in Fig. 4, where lock-in amplifiers 122 and 124 are both locked to modulator 16, and lock-in amplifiers 126 and 128 are both locked to modulator 18.
  • the phase position of amplifier 124 is shifted 90° relatively to the phase position of amplifier 122 and the phase position of amplifier 128 is shifted 90° relatively to that of amplifier 126.
  • amplifiers 122 and 124 constitute a two-phase lock-in amplifier with an in-line and a quadrature channel.
  • amplifiers 126 and 128 also constitute such an amplifier.
  • the outputs I' and I" of a two-phase lock-in amplifier constitute the x and y components of a vector, which, in polar coordinates, represents the magnitude and the phase angle of the signal applied to the amplifier.
  • the transformation to obtain this magnitude and this phase angle from each two-phase lock-in amplifier is performed by the computer 34.
  • phase angle apart from an off-set that can be eliminated by adjustment of the two-phase lock-in amplifier, represents the phase shift between the waveform of the modulated excitation and the waveform of the detected emission.
  • modulation frequencies 2.2 and 3.0 Mhz and with fluorophores that are commonly used for labeling the phase angle shift will be very small, on the order of a few degrees. It will not change substantially from one location of the specimen to another. Therefore, the adjustment of the phase position of a lock-in amplifier to maximize the output is generally quite uncritical. This kind of adjustment has to be performed when using the lock-in amplifiers in the embodiments described earlier. If modulation frequencies are used that are much higher and/or fluorophores are used that have long decay times, substantial phase shifts may appear.
  • phase shift ⁇ A of fluorophore A is derived by the computer 34
  • phase shift ⁇ B of fluorophore B is derived by the computer 34.
  • the magnitude derived by the computer from the outputs of amplifiers 122 and 124 is the measured value of the emission spectrum I A , isolated from other contributions to the total emission in an identical way as described in the specification of the embodiment according to Fig. 1.
  • the magnitude derived by the computer from the outputs of amplifiers 126 and 128 is the measured value of the emission spectrum I B .
  • decay times for each pixel of a scanned image can be calculated and displayed, supplying, in addition to an intensity image of the fluorophore, also a decay time image, alternatively referred to, in the literature, as a "Fluorescence Lifetime Image". This is performed, simultaneously and independently, for two fluorophores, A and B.
  • stored data representing measured values of I A , ⁇ A , I B and ⁇ B are mapped into digital images 72, 73, 78, and 79 for visualization.
  • a schematic block diagram of the second embodiment of the quantitative microfluorometer employing two excitation wavelengths, a single detector and two dual phase lock-in amplifiers is shown.
  • monochromatic excitation light of different wavelengths is supplied by a pair of light sources 12 and 14.
  • the intensity of light source 12 is controlled by a modulator 16 operating at a sinusoidal frequency ⁇ 1
  • light source 14 is controlled by a second modulator 18 operating at a separate frequency ⁇ 2 .
  • modulators 16 and 18 are electro-optical modulators or acousto-optical modulators operating at frequencies up to 50 Mhz.
  • any modulator capable of operating at frequencies preferably at least two times greater than the sampling frequency of the microfluorometer may be substituted.
  • individual light beams from light sources 12 and 14 are combined in beam combiner 20, the combined beam passes through beamsplitter 22 which transmits light at the excitation wavelengths ⁇ 1 and ⁇ 2 , and scanner 24 sequentially directs the combined beam to locations within sample 26.
  • the combined fluorescence emitted in response to excitation at the focal point of scanner 24 is collected by scanner 24 and directed to beamsplitter 22.
  • detector 28 takes the place of detectors 114 and 116 that were used in the dual detector embodiment of Fig. 1.
  • the second beamsplitter 102 is also not used in this embodiment.
  • Detector 28 collects the emitted fluorescence which has been deflected from beamsplitter 22.
  • the signal produced by detector 28 is sent to a pair of lock-in amplifiers 30 and 32.
  • Lock-in amplifier 30 is synchronized with the modulation frequency ⁇ 1 imposed on light source 12 by modulator 16.
  • lock-in amplifier 32 is synchronized with the modulation frequency ⁇ 2 imposed on light source 14 by modulator 18.
  • lock-in amplifiers 30 and 32 are dual phase amplifiers producing two outputs which represent amplitudes of the recorded waveform at phase positions that are 90° apart.
  • the outputs from lock-in amplifiers 30 and 32 are sent to a computer 34.
  • Computer 34 stores the inputs corresponding to each discrete location in sample 26 in a location specific manner and mathematically reconstructs the individual spectral components corresponding to the excitation of each fluorophore by each wavelength.
  • computer 34 is a digital computer and the outputs from amplifiers 30 and 32 are digitized, before storage, by conventional analog to digital converters.
  • ⁇ 1 and ⁇ 2 are selected by examining the absorption spectra 142 and 144 of fluorophores A and B respectively. ⁇ 1 is chosen to predominantly excite fluorophore A, while ⁇ 2 is chosen to predominantly excite fluorophore B. Fluorophore A produces a main fluorescent emission spectrum 146 (I A1 ) in response to ⁇ 1 and a smaller cross-excited fluorescent emission spectrum 148 (I A2 ) in response to ⁇ 2 . This results from the fact that absorption spectra 142 and 144 overlap.
  • fluorophore B produces a main fluorescent emission spectrum 150 (I B2 ) in response to ⁇ 2 and a smaller cross-excited fluorescent emission spectrum 152 (I B1 ) in response to ⁇ 1 .
  • Excitation at wavelength ⁇ 1 produces a combined response 154, which is the sum of fluorophore A main response 146 and fluorophore B cross-excited response 152 (I A1 + I B1 ).
  • excitation at wavelength ⁇ 2 produces a combined response 156 consisting of fluorophore B main response 150 and fluorophore A cross-excited response 148 (I B2 + I A2 ).
  • a combined emission spectrum 140 in response to excitation by ⁇ 1 and ⁇ 2 , is composed of the sum of emission spectra 146, 148, 152 and 150 (I A1 + I A2 + I B1 + I B2 ).
  • Figs. 7a-b the phase relationship between waveforms of the modulated excitation beams and the corresponding emitted fluorescence is shown.
  • the intensity modulation of the beam having excitation wavelength ⁇ 1 is represented by a sinusoidal waveform 160.
  • Fluorophore A responds to ⁇ 1 with a sinusoidally modulated fluorescent main signal 162.
  • Fluorophore B also responds to ⁇ 1 with a smaller modulated fluorescent cross-excited signal 164.
  • the frequencies of excitation signal 160 and fluorescent signals 162 and 164 are the same and will be captured by lock-in amplifier 30 which is synchronized with the modulation frequency ⁇ 1 of ⁇ 1 .
  • the phase of the emitted fluorescence waveform will differ from that of the excitation waveform.
  • the phase relationship between the emitted fluorescence waveform and the excitation waveform is dependent upon the fluorescent relaxation time of the individual fluorophores and on the modulation frequency.
  • Lock-in amplifier 32 is synchronized with the modulation frequency ⁇ 2 of ⁇ 2 .
  • Fluorophore B which is predominantly excited by ⁇ 2 , emits a modulated fluorescent main signal 168 while fluorophore A emits a smaller modulated fluorescent cross-excited signal 170.
  • Fluorescent signals 168 and 170 have the same frequency as excitation 166 and are captured by lock-in amplifier 32. The phases of the emitted fluorescent signals 168 and 170 however will differ from the phase of excitation 166.
  • Figs. 8 and 9 the vector components and phase angles of the in-phase and quadrature channels of lock-in amplifiers 30 and 32, respectively, are shown.
  • the output of the in-phase channel is represented by the quantity I 1 ' and the output of the quadrature channel is represented by the quantity I 1 ".
  • the input to amplifier 30 is represented by a vector of amplitude I 1 at a phase angle V 1 to the in-phase channel. This is composed of the vector sum of the signals corresponding to each fluorophore represented by I A1 at an angle V A1 for fluorophore A and I B1 at angle V B1 for fluorophore B. Similar notation is used in Fig. 9 with the subscript 2 replacing the subscript 1 for the components of lock-in amplifier 32.
  • the following equations are used in computer 34 to resolve the outputs of amplifiers 30 and 32 into the component vectors for fluorophores A and B:
  • phase position of the in-phase channel is tuned to be close to the position that gives a maximum value for the output from the predominantly excited fluorophore.
  • Lock-in amplifier 30 is thus tuned to detect signals from fluorophore A predominantly while lock-in amplifier 32 is tuned to detect signals from fluorophore B predominantly.
  • computer 34 is able to solve the four simultaneous equations (El) having four unknowns, I A1 , I A2 , I B1 and I B2 , using conventional numerical techniques.
  • the derived quantities, I A1 , I A2 , I B1 and I B2 representing the individual components of combined emission spectrum 140 are digitally stored in the memory of computer 34 for each location scanned in specimen 26.
  • the data may be output by computer 34 as displayed images 172, 174, 176 and 178, shown in Fig. 5, corresponding to components I A1 , I B2 , I A2 and I B1 , respectively, or used for further processing.
  • Fig. 10 a schematic block diagram of a variation of the second embodiment of the invention, employing single phase lock-in amplifiers, is shown.
  • the phase position of lock-in amplifier 30, which picks up emission predominantly from fluorophore A is tuned to the position which cancels out the contribution from the waveform generated by fluorophore B.
  • the phase position of lock-in amplifier 32, which picks up emission predominantly from fluorophore B is tuned to the position that cancels out the contribution from the waveform generated by fluorophore A. Regions where one fluorophore is present and the other is not are determined by direct observation.
  • Such regions can be determined by scanning the data set for minimum values of the quotients I 2 /I 1 and I 1 /I 2 , In cases where the fluorophores A and B have different relaxation times, computer 34 is only able to derive the quantities I A1 and I B2 , representing individual components of combined emission spectrum 140.
  • the data may be output by computer 34 as displayed image 172 for I A1 from fluorophore A and 174 for I B2 from fluorophore B.
  • the fluorophores used were TRITC and Lucifer Yellow, at 514 and 458 nm, respectively. These fluorophores were modulated at 0.7 MHz and 0.9 MHz, respectively, using two argon-ion lasers of about 1 watt output power each. The pixel sampling rate was 100 KHz.
  • the ability of the present invention to quantify multiple fluorophores simultaneously may be utilized in a variety of applications. These include the quantitative analysis of fluorescently labeled compounds which have been electrophoretically separated, such as the individual bases in a DNA sequencing. Additionally, the kinetics of reactions involving fluorescently labeled compounds in intact cells may be followed. For example, regions where the fluorescent relaxation times take specific values may be extracted from recorded images. This would open a completely new way to analyze and quantify microscopic structures in three dimensions.

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Claims (24)

  1. Dispositif pour quantifier des cibles fluorescentes par excitation au moyen d'une radiation électromagnétique présentant de multiples longueurs d'onde discrètes, comprenant :
    des moyens pour diriger des rayons doubles de radiation électromagnétique continue, chaque rayon présentant une longueur d'onde discrète, cependant que lesdits rayons se rencontrent de manière à produire un balayage en une pluralité d'endroits individuels où des cibles fluorescentes sont présentes, en provoquant l'excitation de celles-ci ;
    des moyens pour moduler l'intensité de chacun desdits rayons à des fréquences de modulation séparées pour donner à chacun desdits rayons une forme d'onde spécifique modulée dans le temps ;
    des moyens pour détecter l'émission fluorescente combinée provenant desdites cibles en réponse à ladite excitation ; et :
    des moyens de démodulation comprenant au moins deux amplificateurs de blocage, chaque amplificateur étant associé à une fréquence de modulation différente, pour extraire de ladite émission fluorescente combinée détectée les contributions qui correspondent à chacune desdites cibles, en quantifiant ainsi lesdites cibles.
  2. Dispositif selon la revendication 1, dans lequel lesdits moyens pour détecter l'émission fluorescente combinée provenant desdites cibles comprennent des détecteurs doubles.
  3. Dispositif selon la revendication 1, dans lequel lesdits moyens pour détecter l'émission fluorescente combinée provenant desdites cibles comprennent un détecteur unique.
  4. Dispositif pour quantifier des cibles fluorescentes distribuées dans un plan de l'espace par excitation au moyen d'une radiation électromagnétique présentant de multiples longueurs d'onde discrètes, comprenant :
    des moyens pour diriger des rayons doubles de radiation électromagnétique présentant des longueurs d'onde discrètes différentes vers une pluralité d'endroits individuels de manière à produire un balayage dans un plan où des cibles fluorescentes sont présentes, en provoquant l'excitation des cibles ;
    des moyens pour moduler chacun desdits rayons à des fréquences de modulation séparées pour donner à chacun desdits rayons une forme d'onde spécifique modulée dans le temps ;
    des moyens pour recueillir la radiation émise en des endroits dudit plan en réponse à ladite excitation ;
    des moyens pour séparer optiquement des parties du spectre de ladite radiation émise, lesdites parties du spectre représentant au moins deux bandes de longueurs d'onde différentes ;
    des moyens pour détecter indépendamment la radiation qui représente chacune desdites parties séparées du spectre de ladite radiation émise ;
    des moyens de démodulation comprenant au moins deux amplificateurs de blocage, chaque amplificateur étant associé à une fréquence de modulation différente, pour extraire de chacune desdites parties détectées du spectre de la radiation combinée émise depuis des cibles fluorescentes multiples les contributions qui correspondent à chacune desdites cibles, en quantifiant ainsi chaque cible dans ledit plan.
  5. Dispositif selon la revendication 4, dans lequel chacun desdits rayons est produit par une diode laser séparée, chacune desdites diodes laser étant modulée en intensité à une fréquence séparée.
  6. Dispositif selon la revendication 4, dans lequel lesdits moyens pour moduler chacun desdits rayons comprennent un modulateur optoélectrique pour chaque rayon, chacun des rayons étant modulé à une fréquence séparée.
  7. Dispositif selon la revendication 4, dans lequel lesdits moyens pour moduler chacun desdits rayons comprennent un modulateur optoacoustique pour chaque rayon, chacun des rayons étant modulé à une fréquence séparée.
  8. Dispositif selon la revendication 4, dans lequel lesdits moyens pour détecter indépendamment des parties du spectre de ladite émission combinée comprennent des détecteurs doubles, et lesdits moyens pour séparer optiquement des parties du spectre comprennent des filtres optiques susceptibles de distribuer à chacun desdits détecteurs doubles des parties de spectre séparées de ladite émission fluorescente combinée.
  9. Dispositif selon la revendication 8, dans lequel lesdits moyens de démodulation comprennent des moyens de démodulation séparés pour chacun desdits détecteurs doubles, lesdits moyens de démodulation étant propres à des signaux présentant les mêmes fréquences que la fréquence de modulation d'un rayon correspondant, et le signal de sortie de chaque moyen de démodulation étant propre à une position particulière de la phase de la forme d'onde modulée.
  10. Dispositif selon la revendication 9, dans lequel lesdits moyens de démodulation comprennent un amplificateur de blocage à phase unique pour chacun des détecteurs doubles, ledit amplificateur étant synchronisé avec la fréquence de modulation du rayon correspondant.
  11. Dispositif selon la revendication 10, dans lequel la position de la phase de chacun desdits amplificateurs à phase unique est accordée sur la position qui rend maximal le signal de sortie dudit amplificateur provenant d'une cible fluorescente correspondante.
  12. Dispositif selon la revendication 9, dans lequel lesdits moyens de démodulation comprennent deux amplificateurs de blocage à phase unique pour chacun desdits détecteurs doubles, l'un étant synchronisé avec la fréquence de modulation de l'un desdits rayons d'excitation et l'autre l'étant avec la fréquence de modulation de l'autre, des spectres à excitation croisée étant mesurés simultanément et indépendamment.
  13. Dispositif selon la revendication 9, dans lequel lesdits moyens de démodulation comprennent un amplificateur de blocage à phase double pour chacun desdits détecteurs doubles, des déphasages de deux cibles fluorescentes étant mesurés simultanément et indépendamment.
  14. Dispositif selon la revendication 4, dans lequel lesdits moyens pour recueillir la radiation émise en des endroits dudit plan est un microscope à foyer commun.
  15. Dispositif pour quantifier des cibles fluorescentes distribuées dans un volume de l'espace par excitation au moyen d'une radiation électromagnétique présentant de multiples longueurs d'onde discrètes, comprenant :
    des moyens pour diriger des rayons doubles de radiation électromagnétique présentant des longueurs d'onde discrètes différentes vers une pluralité d'endroits individuels de manière à produire un balayage sur un plan de balayage d'un échantillon volumique où une ou plusieurs cibles fluorescentes sont présentes, en provoquant l'excitation de la cible ;
    des moyens pour moduler chacun desdits rayons à des fréquences de modulation séparées pour donner à chacun desdits rayons une forme d'onde spécifique modulée dans le temps ;
    des moyens à foyer commun pour recueillir la radiation émise en des endroits du plan de balayage en réponse à ladite excitation ;
    des moyens pour séparer optiquement des parties du spectre de ladite radiation émise, lesdites parties du spectre représentant au moins deux bandes de longueurs d'onde différentes ;
    des moyens pour détecter indépendamment la radiation qui représente chacune desdites parties séparées du spectre de ladite radiation émise ;
    des moyens de démodulation comprenant au moins deux amplificateurs de blocage, chaque amplificateur étant associé à une fréquence de modulation différente, pour extraire de chacune desdites parties détectées du spectre de la radiation combinée émise depuis des cibles fluorescentes multiples les contributions qui correspondent à chacune desdites cibles, en quantifiant ainsi chaque cible sur le plan de balayage ;
    des moyens pour remplacer le plan de balayage par un autre plan de balayage de l'échantillon volumique ; et :
    des moyens pour mettre en mémoire des données pour une pluralité de plans de balayage représentant la quantification de cibles fluorescentes dans un échantillon volumique.
  16. Dispositif selon la revendication 15, dans lequel chacun desdits rayons est produit par une diode laser séparée, chacune desdites diodes laser étant modulée à une fréquence séparée.
  17. Dispositif selon la revendication 15, dans lequel lesdits moyens pour moduler chacun desdits rayons comprennent un modulateur optoélectrique pour chaque rayon.
  18. Dispositif selon la revendication 15, dans lequel lesdits moyens pour moduler chacun desdits rayons comprennent un modulateur optoacoustique pour chaque rayon.
  19. Dispositif selon la revendication 15, dans lequel lesdits moyens pour détecter indépendamment des parties du spectre de ladite émission combinée comprennent des détecteurs doubles, et lesdits moyens pour séparer optiquement les parties du spectre comprennent des filtres optiques susceptibles de distribuer à chacun desdits détecteurs doubles des parties de spectre séparées de ladite émission fluorescente combinée.
  20. Dispositif selon la revendication 19, dans lequel lesdits moyens de démodulation comprennent des moyens de démodulation séparés pour chacun desdits détecteurs doubles, lesdits moyens de démodulation étant propres à des signaux présentant les mêmes fréquences que la fréquence de modulation d'un rayon correspondant, et le signal de sortie de chaque moyen de démodulation étant propre à une position particulière de la phase de la forme d'onde modulée.
  21. Dispositif selon la revendication 20, dans lequel lesdits moyens de démodulation comprennent un amplificateur de blocage à phase unique pour chacun des détecteurs doubles, ledit amplificateur étant synchronisé avec la fréquence de modulation du rayon correspondant.
  22. Dispositif selon la revendication 21, dans lequel la position de la phase de chacun desdits amplificateurs à phase unique est accordée sur la position qui rend maximal le signal de sortie dudit amplificateur provenant d'une cible fluorescente correspondante.
  23. Dispositif selon la revendication 20, dans lequel lesdits moyens de démodulation comprennent deux amplificateurs de blocage à phase unique pour chacun desdits détecteurs doubles, l'un étant synchronisé avec la fréquence de modulation de l'un desdits rayons d'excitation et l'autre l'étant avec la fréquence de modulation de l'autre, des spectres à excitation croisée étant mesurés simultanément et indépendamment.
  24. Dispositif selon la revendication 20, dans lequel lesdits moyens de démodulation comprennent un amplificateur de blocage à phase double pour chacun desdits détecteurs doubles, des déphasages de deux cibles fluorescentes étant mesurés simultanément et indépendamment.
EP94906355A 1993-02-01 1994-02-01 Appareil d'imagerie quantitative de fluorogenes multiples Expired - Lifetime EP0681695B1 (fr)

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US08/011,881 US5294799A (en) 1993-02-01 1993-02-01 Apparatus for quantitative imaging of multiple fluorophores
US11881 1993-02-01
US189190 1994-01-31
US08/189,190 US5418371A (en) 1993-02-01 1994-01-31 Apparatus for quantitative imaging of multiple fluorophores using dual detectors
PCT/IB1994/000019 WO1994018547A1 (fr) 1993-02-01 1994-02-01 Appareil d'imagerie quantitative de fluorogenes multiples

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US5418371A (en) 1995-05-23
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DE69402958D1 (de) 1997-06-05
DE69402958T2 (de) 1997-12-11
ATE152519T1 (de) 1997-05-15

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